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Geretti AM, editor. Antiretroviral Resistance in Clinical Practice. London: Mediscript; 2006.

Chapter 4 Resistance to entry inhibitors

Authors

Eva Poveda and Vincent Soriano.

Introduction
Viral entry is currently one of the most important targets in the search for new drugs to treat HIV-1 infection. Advances
in the knowledge of the molecular mechanisms involved in the different stages of the entry process have allowed the
production of molecules that block each step: (1) attachment of the viral glycoprotein gp120 to the CD4 cell receptor;
(2) binding of the gp120 to chemokine co-receptors CCR5 or CXCR4; and (3) the gp41-mediated fusion of the viral
and cellular membranes. Entry inhibitors are the latest family of antiretroviral compounds to come into clinical use.
The first compound to be approved was the fusion inhibitor enfuvirtide [1–3]. Many other entry inhibitors are currently
in clinical development and it is hoped that they will soon be part of the therapeutic armamentarium against HIV-1.
This new family of antiretroviral agents is eagerly awaited by the growing number of patients carrying drug-resistant
viruses to reverse transcriptase and protease inhibitors. However, clinical experience has taught us that HIV-1 almost
always finds a way to escape, mutating and evading drug-selective pressure [4]. In this chapter, in vitro and clinical
data concerning the mechanisms of resistance to entry inhibitors will be reviewed (Table 1).

Resistance to CD4–gp120 binding inhibitors

The first step in the viral entry process is the attachment of the viral gp120 to the CD4 receptor present in the cell
surface. It is mainly driven by electrostatic forces between the positive charge of the CD4 molecule and the negative
charge of the gp120 pocket. Van der Waals' forces and hydrogen bonds help to stabilise the initial CD4–gp120
interaction. It is estimated that the amino acid Phe 43 in the CD4 receptor accounts for 23% of the binding affinity to
HIV-1 gp120 [5]. Following CD4–gp120 binding, gp120 undergoes conformational changes that allow the subsequent
interaction with CCR5 or CXCR4 on the cell surface.

There are many candidate inhibitors of CD4–gp120 binding, including PRO-542 [CD4-immunoglobulin G2 (IgG2)],
TNX-355, BMS-806 and CADA, each characterised by a different structure and mechanism of action [6–8].
Information on resistance to these inhibitors is scarce. In vitro studies of BMS-806 and related compounds (e.g. BMS-
155) have shown that the amino acids of gp120 implicated in resistance are those surrounding the Phe 43 pocket.
Changes in five gp120 residues (Trp 112, Thr 257, Ser 375, Phe 382 and Met 426) result in escape from inhibition by
BMS-806 and BMS-155. The degree of sequence conservation of the nearby V1/V2 variable loops indirectly
influences the sensitivity to these drugs [9]. Natural gp120 variability among different HIV-1 subtypes may account for
differences in baseline susceptibility to this class of compounds. This is the case for subtype C and recombinant
subtype AE (CRF01_AE), which seem to be naturally resistant to BMS-806 [10].

Resistance to CCR5 and CXCR4 antagonists

In the second step of viral entry, the CD4–gp120 interaction provokes conformational changes in the viral envelope
that allow the CD4–gp120 complex to interact with the CCR5 or CXCR4 chemokine co-receptor. The V3 loop in HIV-
1 gp120 is the main domain involved in this interaction and V3 amino acid sequences largely determine the preferential
use of CCR5 or CXCR4 by HIV-1 as a co-receptor for entry into the cells [11]. Accordingly, HIV-1 isolates may be
classified as CCR5 tropic (R5), CXCR4 tropic (X4) or dual tropic R5/X4 strains, depending on their co-receptor use
[12].

Several compounds designed to block the CCR5–gp120 interaction of R5 strains are currently in clinical development,
including SCH-C, vicriviroc (SHC-D), aplaviroc (GW873140), maraviroc (UK-427,857), TAK-220, TAK-652 and
PRO-140. The sites of interaction of these molecules with CCR5 have been mapped within the pocket formed by the
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transmembrane helices of CCR5 [13]. The binding of CCR5 antagonists with the co-receptor disrupts the conformation
of CCR5, specifically the second extracellular loop (ECL2), which can no longer interact with the V3 loop of HIV-1
gp120 [14,15]. Some inhibitors, such as aplaviroc, appear to interact directly with ECL2 [16]. The clinical development
of this compound, however, has recently been halted (at least in drug-naive patients), due to the development of liver
toxicity in some patients [17].

Two main resistance pathways have been described for HIV-1 to escape from CCR5 or CXCR4 antagonists. The first is
a shift in co-receptor usage. The second results from changes in regions of the viral envelope that allow the interaction
between gp120 and the co-receptor to occur despite the presence of the inhibitor. An increased affinity of gp120 for
CCR5 has been shown to enable more efficient competition of HIV-1 with CCR5 inhibitors for binding to the CCR5
co-receptor [18]. Data available to date suggest that most CCR5 antagonist-resistant strains preserve the use of the
CCR5 co-receptor, and that multiple mutations within different regions of gp120 [V3, V2 and constant regions 2 and 4
(C2 and C4)] account for the drug-resistant phenotype [19–21]. It should be noted that most mutations are specific for
each compound, which may limit cross-resistance and allow sequencing within this class of compounds. However,
large clinical studies are needed to confirm these in vitro observations. Preliminary findings with viral isolates resistant
to maraviroc have demonstrated that they remain susceptible to SCH-C, vicriviroc and aplaviroc [22]. In contrast,
vicriviroc-resistant strains show cross-resistance to SCH-C, AD101 and regulation on activation, normal T cell
expressed and secreted (RANTES) derivatives, most probably because they share the site of interaction with CCR5
[21]. It is clear that CCR5 antagonist-resistant strains do not show cross-resistance to the currently approved reverse
transcriptase and protease inhibitors. In addition, there is no evidence of cross-resistance with other classes of entry
inhibitors, including CD4–gp120 binding inhibitors and enfuvirtide [21].

CXCR4 antagonists block the interaction of the CD4–gp120 complex with the ECL2 domain of the chemokine co-
receptor CXCR4 [23–25]. Some of these compounds, such as AMD-070, KRH-1636 and KRH-2731, are currently in
pre-clinical and clinical development [8], but information on selection of resistance is scarce. Mutations in the V3
domain seem to account for the loss of susceptibility to many of these drugs. However, changes in other gp120 regions
(V1, V2 and V4) have also been observed, including deletion of five amino acids (364–368) within the V4 domain
[26,27].

Preliminary results have failed to identify a shift in co-receptor use as the main resistance pathway for evading CXCR4
antagonists; however, it will be necessary to wait for the results of ongoing larger clinical trials to confirm this. It
should be noted that while the demonstration of a shift in co-receptor use from CCR5 towards CXCR4 could accelerate
HIV-1 disease progression, the contrary could be the case using CXCR4 antagonists, given that R5 viruses tend to be
associated with less virulent HIV-1 disease. It is worth noting that recent data suggest that the consideration of a
patient's virus population as R5 or X4 is too simple. Most subjects carry a predominant virus population with the other
present as minor quasispecies. Under treatment with CCR5 antagonists, replacement of R5 by X4 viruses could occur
as the result of drug pressure. In most cases, the virus population emerging as predominant was present already,
although it represented a low proportion of the total virus population.

Resistance to fusion inhibitors

Following the interaction between viral gp120 and the cell CD4 receptor, and the interaction with chemokine co-
receptors, another conformational change occurs in the viral envelope that causes a shift from a non-fusogenic to a
fusogenic state. The viral gp41 protein, which comprises the repeat regions 1 (HR1) and 2 (HR2), drives the fusion
process. The N-terminus domain of gp41 is exposed and inserted through the fusion peptide (FP) into the cellular
membrane, allowing viral and cellular membrane fusion. Thereafter, the viral capsid enters the cytoplasm [3,28].

Enfuvirtide, the first entry inhibitor to be approved for clinical use, is a synthetic peptide that mimics the HR2 fragment
of gp41. Its binding to the HR1 region blocks the formation of a six-helix bundle structure, which is crucial for the
fusion process. Early in vitro studies showed that resistance to enfuvirtide involved the selection of changes in three
amino acids (positions 36–38) within the HR1 region of gp41 [29]. However, further research has revealed that
virological failure in patients receiving enfuvirtide therapy may involve the selection of changes in a larger fragment of
HR1, from codons 36 to 45 [30–32] (Figure 1). As expected, the impact on the loss of susceptibility to enfuvirtide
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caused by changes at different positions varies considerably, although it appears that changes in simple amino acids
may be responsible for high-level resistance to enfuvirtide in some cases [33]. Therefore, enfuvirtide should be
considered as a drug with low genetic barrier for resistance. The clinical implication of this fact is that the optimal use
of the drug is in combination with other active antiretroviral compounds and never as the only active agent in a
regimen.

There is a wide range of susceptibility to enfuvirtide in viral isolates collected from enfuvirtide-naive patients, as well
as from individuals undergoing enfuvirtide therapy and harbouring the same resistance mutations [31,33,34]. The
determinants of this broad heterogeneity in virus susceptibility are unclear, but naturally occurring polymorphisms in
the HR2 region of gp41 as well as changes in HR2 selected during therapy with enfuvirtide may explain this
phenomenon [35]. Changes in HR2 have been observed in patients receiving long-term enfuvirtide therapy, although
they do not seem to follow a consistent pattern. Therefore, at present, it is difficult to conclude how changes in HR2
influence susceptibility to enfuvirtide [33,36]. In addition to being directly implicated in enhancing resistance, changes
in HR2 may also act as compensatory mutations, thus improving viral fitness [37].

Controversy exists regarding the impact of HIV-1 co-receptor tropism on the susceptibility to enfuvirtide. Although
some in vitro studies have shown that R5 strains are resistant to enfuvirtide [38,39], in vivo studies have not found
significant differences in response to enfuvirtide therapy when comparing patients harbouring R5 or X4 strains [40,41].

In addition to viral factors, host determinants may also influence the susceptibility to enfuvirtide. A relationship
between the level of co-receptor expression on target cells and fusion kinetics has been found, such that the presence of
high levels of CCR5 on the cellular surface results in more rapid membrane fusion, reducing the time in which gp41
could be targeted by enfuvirtide. Thus, individuals carrying delta-32 deletions in the CCR5 co-receptor (Δ32-CCR5),
who express low levels of CCR5, might consequently respond more favourably to enfuvirtide [39].

Conclusions
Entry inhibitors mark the beginning of a new era in the history of antiretroviral therapy, opening new therapeutic
options for the already large and growing number of patients carrying drug-resistant viruses. Enfuvirtide is the first
agent of this class approved for clinical use. Several other compounds are currently in clinical development and may
soon be available for use in the treatment of HIV-1. Available evidence indicates that selection of drug resistance may
occur with these compounds. However, the pathways leading to resistance to entry inhibitors differ substantially from
those causing resistance to the antiretrovirals in current use, and therefore no cross-resistance is anticipated between
entry inhibitors and other classes of antiretrovirals, thus allowing salvage therapy with entry inhibitors.

The main mechanism of resistance to enfuvirtide is the selection of changes in a domain consisting of 10 amino acids,
between residues 36 and 45 in the HR1 region of gp41. For other entry inhibitors, multiple changes in different gp120
domains (V3, C2, C4 and V4) seem to be responsible for causing loss of susceptibility, although with limited cross-
resistance in most cases. Finally, natural susceptibility of different HIV-1 variants to entry inhibitors warrants further
investigation, given that most entry inhibitors target the most variable HIV-1 proteins.

References
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35. Stanfield-Oakley S, Jefrey J, McDanal C. et al. Determinants of susceptibility to enfuvirtide map to gp41 in
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36. Xu L, Pozniak A, Wildfire A et al. Evolution of HR1 and HR2 mutations in HIV-1 gp41 associated with long-
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37. Cabrera C, Garc'a E, Marfil S et al. Resistance to enfuvirtide (ENF) proceeds through selection of mutations in
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38. Derdeyn C, Decker J, Sfakianos J. et al. Sensitivity of HIV type 1 to fusion inhibitors targeted to the gp41 first
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40. Whitcomb J, Huang W, Fransen S et al. Analysis of baseline enfuvirtide (T-20) susceptibility and co-receptor
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Figures

Figure 1
The gp41 lineal structure and enfuvirtide sequences that mimic HR2. FP, fusion peptide; CC, cysteine–cysteine; tm,
transmembrane domain; NH, N-terminal.

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Tables

Table 1 HIV entry inhibitors: mechanisms of action and possible resistance pathways

Drug Mechansim of action Resistance pathway

CD4–gp120 inhibitors
PRO-542 Tetravalent recombinant antibody (CD4-IgG2) Changes within gp120 surrounding
the Phe 43 cavity
TNX-355 Monoclonal antibody against CD4
BMS-806 Binds to gp120 blocking conformational changes after CD4
binding
CADA Decreases CD4 expression on the cell surface
CCR5 antagonists
SCH-C, SCH-D Binds to transmembrane domains of CCR5 Changes within gp120 (V3, C2, V2
and C4)
GW873140 Binds to ECL2 of CCR5 Co-receptor shift to CXCR4 use
Maraviroc (UK- Binds to transmembrane domains of CCR5
427,857)
TAK-220, Binds to transmembrane domains of CCR5
TAK-652
PRO-140 Monoclonal antibody against CCR5
CXCR4 antagonists
AMD070 Binds to CXCR4 Changes within gp120 (V3, V1,
V2 and V4)
KRH-1636, Binds to second and third extracellular loops of CXCR4 Co-receptor shift to CCR5 use
KRH-2731
Fusion inhibitors
Enfuvirtide (T- Synthetic peptide that mimics an HR2 fragment and blocks the Changes at residues 36–45 in the
20)* formation of the six-helix bundle HR1 region of gp41

* Approved by the US Food and Drug Administration for clinical use since 2003.

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Bookshelf ID: NBK2251 PMID: 21249775

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